Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental defects caused by prenatal alcohol (ethanol) exposure. FASD has great variation in clinical manifestations, such as physical, mental and growth retardation, and defects of the heart, kidney, muscle, skeleton, eyes, ears, and other craniofacial regions. Although the underlying physiology and biochemistry of all cells is similar, studies have shown that fetal cells are more sensitive to ethanol than adult cells, and the in vivo and in vitro effects of ethanol may vary from induction of apoptosis to the inhibition of proliferation, differentiation, migration or other function. The effect of ethanol on stern cells remain unclear. Stem cells have the ability to self-renew and differentiate into multiple cell types. Stem cells maybe used as a tool elucidate mechanisms of ethanol's effect on organogenesis. For the current proposal, we plan to use a particular type of stem cells, amniotic fluid-derived stem cells (AFSC), as a model system. AFSC are multi-potent stem cells that express embryonic stem cell markers, differentiate in vivo into all 3 germ layers when injected into blastocysts, and have the capacity to differentiate in vitro into bone, muscle, fat, endothelium, liver, and neurons. Because this cell type has the capacity to differentiate into numerous type of cells, it provides an intriguing new model system with which to analyze potential mechanisms underlying the genesis of FASD. We hypothesize that in utero ethanol exposure will decrease the number of AFSC in the amniotic fluid and decrease the efficiency of differentiation of AFSC. In order to address this hypothesis, we propose to meet the following specific aims. Aim 1: To characterize the effects of in utero ethanol exposure on AFSC number and their growth potential. Aim 2: To determine the effects of in utero ethanol exposure on AFSC differentiation and function. Although these cell are not part of the embryo, AFSC appear to have the potential to serve as an excellent model system for discovering the effects of ethanol on cellular processes, in particular, they will provide a unique opportunity to examine the effects of ethanol on stem cell differentiation to multiple cell types. Achieving the goals of this study will provide new information about mechanism underlying the early teratogenic effects of ethanol on stem cell biology and provide insight into ethanol's effect on the developing embryo.